Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Portions of closed jejunal biopsies from the dog were homogenised and their organelles separated by isopycnic centrifugation on continuous sucrose density gradients. The distributions of marker enzymes for the principal organelles were determined using highly sensitive assay procedures. The following organelles, with assayed marker enzymes and modal densities between brackets were characterised: peroxisomes (catalase, 1.21); brush borders (zinc-resistant alpha-glucosidase, leucyl-beta-naphthyl-amidase, gamma-glutamyl transferase, alkaline phosphatase, 1.20); lysosomes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, 1.19); mitochondria (malate dehydrogenase, 1.18); endoplasmic reticulum (Tris-resistant alpha-glucosidase, 1.16); basal-lateral membranes (5'-nucleotidase, 1.11) and cytosol (lactate dehydrogenase). Homogenisation in isotonic sucrose containing digitonin (0.12 mmol/litre) selectively disrupted lysosomes and increased the equilibrium density of brush border and basal-lateral membranes. This procedure will be used to study the subcellular pathology of naturally occurring intestinal disease in the dog.
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PMID:Subcellular fractionation studies on peroral jejunal biopsies from the dog. 3 Jan 25

Transfer of truncated oligosaccharides to protein in vivo and the structure of Man2GlcNAc2 synthesized by intact yeast (Saccharomyces cerevisiae) were investigated in the alg2 mutant. At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man2GlcNAc2 and Man1GlcNAc2 (T.C. Huffaker and P.W. Robbins (1983) Proc. Natl. Acad. Sci. USA 80, 7466-7470). We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC. Man2GlcNAc2 and Man1GlcNAc2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) dependent. Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype. The alg2/pAC3 cells are viable at 36 degrees C. Two isomers of Man2GlcNAc2, Man1----3ManGlcNAc2 and Man1----6ManGlcNAc2, were present on lipid and protein. The transfer of Man2GlcNAc2 and Man1GlcNAc2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.
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PMID:Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man2GlcNAc2 and Man1GlcNAc2 are transferred from dolichol to protein in vivo. 266 Jul 43

Considerable evidence indicates that the connecting tubule cells, a type of cell of the distal nephron which seems to participate on potassium secretion, may be the place where renal kallikrein is synthetized. As potassium secretion and kallikrein synthesis may occur in the same cells, we studied the effect of high potassium diet on renal kallikrein production. The kallikrein containing cells from rats fed a normal and high potassium diet were evaluated using a combination of morphometric analysis, conventional electron microscopy, and ultrastructural immunocytochemistry. High potassium diet produced hypertrophy and hyperplasia of the kallikrein containing cells. Hyperplasia was sustained by an increased number of immunoreactive cells/mm2 (151 +/- 14 vs. 86.4 +/- 12, P less than 0.01), an increased number of binucleated immunoreactive cells/mm2 (11.90 +/- 2.1 vs. 3.77 +/- 0.17, P less than 0.005), and by the presence of mitosis. Cell hypertrophy was sustained by an increased cross-sectional area of immunoreactive cells (mu 2) (320.4 +/- 21 vs. 104.5 +/- 6.1, P less than 0.001), by an increased area of basal plasma membrane infoldings, by an hypertrophy of the components of the Golgi complex, hypertrophy of the components of the rough endoplasmic reticulum, and by a larger number of secretory-like vesicles containing kallikrein. The rats fed with high potassium diet had higher values on urinary kallikrein excretion-amidase activity (3.70 +/- 0.51 vs. 2.01 +/-0.37 units/day, P less than 0.02), higher values on potassium excretion (18.8 +/- 1.7 vs. 1.31 +/- 0.1 mmol/day, P less than 0.001), and higher urinary volume (51.5 +/- 5.3 vs. 12.2 +/- 1.6 ml/day, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for a stimulatory effect of high potassium diet on renal kallikrein. 330 6

Aspartylglucosaminidase (AGA, E.C. 3.5.1.26) is a soluble lysosomal hydrolase that participates in the degradation of glycoproteins. Here we analyzed the special features in the intracellular targeting of this dimeric amidohydrolase, especially the role of N-linked sugars and their phosphorylation in transport and activity of heterodimeric aspartylglucosaminidase, using in vitro mutagenesis and transient expression of mutant polypeptides in COS cells. The single N-glycosylation sites of both the alpha and beta subunits were destroyed individually and in combination. Just one remaining N-glycosylation site on either subunit was sufficient for normal processing into subunits and lysosomal transport, but the totally nonglycosylated enzyme, although active and processed into subunits, was not transported into lysosomes and became trapped in the endoplasmic reticulum (ER) or secreted. The intracellular targeting of AGA was partially disturbed by the lack of glycosylation in the beta subunit, resulting in accumulation of dimeric, active polypeptides in the ER, whereas lack of oligosaccharides in the alpha subunit did not affect the intracellular targeting of AGA. N-glycans in the beta subunit were found to be essential for the long-term stability of the polypeptide in the cell, but not for initial folding or subunit processing into the active dimeric molecule. Both subunits have two glycosylation isoforms. Both forms of the alpha subunit were found to be phosphorylated, whereas only one of the two glycosylation isoforms of the beta subunit is phosphorylated. The mutant enzyme with nonglycosylated alpha subunit and nonphosphorylated beta subunit is transported into lysosomes, suggesting that AGA is capable of using an alternative, mannose-6-phosphate receptor-independent routing into lysosomes.
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PMID:Intracellular sorting of aspartylglucosaminidase: the role of N-linked oligosaccharides and evidence of Man-6-P-independent lysosomal targeting. 771 Jun 87

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a dimeric lysosomal hydrolase involved in the degradation of glycoproteins. The synthesized precursor polypeptide of AGA is rapidly activated in the endoplasmic reticulum by proteolysis into two subunits. Expression of the alpha- and beta-subunits of AGA in separate cDNA constructs showed that independently folded subunits totally lack enzyme activity, and even when co-expressed in vitro they fail to produce an active heterodimer of the enzyme. Both of the subunits are required for the enzyme activity, and the immediate interaction of the subunits in the endoplasmic reticulum is necessary for the correct folding of the dimeric enzyme molecule. The specific amino acid residues essential for the active site of the AGA enzyme were further analyzed by site-directed mutagenesis and in vitro expression of mutagenized constructs. Replacement of Thr206, the most amino-terminal residue of the beta-subunit, with Ser resulted in a complete loss of enzyme activity without influencing intracellular processing or transport of the mutant polypeptide to the lysosomes. Analogously, replacement of the most amino-terminal tryptophan, Trp34 with Phe or Ser in the alpha-subunit, resulted in a totally inactive enzyme without influencing the intracellular processing or stability of the polypeptide. These results suggest that the catalytic center of this amidase is formed by the interaction of the amino-terminal parts of two subunits and requires both Trp34 in the alpha-subunit and Thr206 in the beta-subunit.
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PMID:Immediate interaction between the nascent subunits and two conserved amino acids Trp34 and Thr206 are needed for the catalytic activity of aspartylglucosaminidase. 787 64

Oligosaccharides on invertase restricted to the endoplasmic reticulum (ER) in alg3,sec18 yeast at 37 degrees C were found to be 20% wild type Man8GlcNAc and 80% Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). These results suggested that alg3 was slightly leaky, but did not address whether the oligosaccharide-lipid Man9GlcNAc2 and Man5GlcNAc2 precursors were glucosylated in alg3 yeast. Therefore, an alg3,sec18,gls1 strain was constructed to delete the GLS1-encoded glucosidase I responsible for trimming the terminal alpha 1,2-linked glucose from newly transferred Glc3ManxGlcNAc2 oligosaccharides. Invertase activity was overexpressed 5-10-fold on transforming this strain with a multicopy plasmid (pRB58) carrying the SUC2 gene, and preparative amounts of the ER form of external invertase, derepressed and accumulated at 37 degrees C, were purified. The N-linked glycans were released by sequential treatment with endo-beta-N-acetylglucosaminidase H (endo H) and peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase. Oligosaccharide pools were sized separately on Bio-Gel P-4, which showed that endo H released about 17% of the carbohydrate as Glc3Man8GlcNAc, while peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase released the remainder as Hex8GlcNAc2 and Man5GlcNAc2 in a 1:4 ratio. Glycan structures were assigned by 500-MHz two-dimensional DQF-COSY 1H NMR spectroscopy, which revealed that the endo H-resistant Hex8GlcNAc2 pool contained Glc3Man5GlcNAc2 and Man8GlcNAc2 in a 6:4 ratio, the latter a different isomer from that formed by the ER alpha 1,2-mannosidase (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). Recovery of Glc3Man8GlcNAc and not the ER form of Man8GlcNAc provided an internal control indicating the absence of glucosidase I, which was confirmed by incubation of [3H]Glc3[14C]Man9GlcNAc with solubilized membranes from either alg3,sec18,gls1 or alg3,sec18,GLS1 strains. Chromatographic analysis of the products showed that [3H]Glc was removed only in the presence of the GLS1 gene product. Thus, the vast majority of the N-linked glycosylation in the ER of alg3 yeast (> 75%) occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally found on the lipid-linked precursor.
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PMID:Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. I. Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum. 850 33

Alg3 yeast mutants synthesize endoglycosidase H-resistant oligosaccharides whose precursor for elongation is Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R. B. (1991) J. Biol. Chem. 266, 5547-5551). To characterize alg3 glycan elongation in vivo, oligosaccharides on alg3,sec18 invertase synthesized and secreted at 26 degrees C were released with peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase and purified by Bio-Gel P-4 chromatography. Large (Man > 30GlcNAc2) and intermediate (Man5-10GlcNAc2) sized oligosaccharides were pooled separately, and the smaller ones were exchanged with 2H2O for one- and two-dimensional DQF-COSY 1H NMR analyses at 500 MHz. Although there was no detectable substitution of the terminal alpha 1,6-core-linked mannose, addition of alpha 1,6-, alpha 1,2-, and alpha 1,3-mannoses to the alpha 1,3-linked core branch of a majority of the Man5 precursor was analogous to core-filling reactions seen on wild type invertase glycans (Trimble, R.B., and Atkinson, P.H. (1986) J. Biol. Chem. 261, 9815-9824). Two additional types of oligosaccharide structures were found; those which retained glucose and those consistent with mannan elongation. Glucose retention appeared to be due to inefficient trimming from minor glucosylated intermediates, while mannan elongation was by extension of a new alpha 1,6-linked branch from the alpha 1,3-core-linked residue as seen in wild-type core oligosaccharides (Hernandez, L.M., Ballou, L., Alvarado, E., Gillece-Castro, B.L., Burlingame, A.L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856) or mnn1,mnn2,mnn10 processing intermediates (Ballou, L., Alvarado, E., Tsai, P-k., Dell, A., and Ballou, C.E. (1989) J. Biol. Chem. 264, 11857-11864). Thus, the alpha 1,6-linked branch additions which form Man9GlcNAc2-PP-dolichol from Man5GlcNAc2-PP-dolichol appear to provide important structural information enabling efficient recognition by the endoplasmic reticulum-glucosyltransferases forming oligosaccharide-lipid as well as the glucosidases involved in early trimming reactions, but the alg3 mutant documents that they are unnecessary for normal yeast mannan elongation.
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PMID:Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant. II. Structure of novel Man6-10GlcNAc2 processing intermediates on secreted invertase. 850 34

Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N4-N-acetyl-beta-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc2 can be transferred from dolichol-PP-GlcNAc2 to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum.
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PMID:N-glycosylation by transfer of GlcNAc2 from dolichol-PP-GlcNAc2 to the protein moiety of the major yeast exoglucanase. 967 21

Fatty acid amide hydrolase contains a proline-rich sequence matching a consensus sequence for SH3-binding domains as well as a transmembrane domain. In this study, deletion mutants lacking the proline-rich region and the transmembrane domain were generated. Transfection experiments demonstrated that the proline-rich deleted amidase was enzymatically inactive. While immunostaining of the wild-type was always punctate with strong perinuclear staining characteristic for endoplasmic reticulum, the staining of the mutant was diffuse and distributed throughout the cytoplasm and perinuclear region. These observations along with the loss of activity suggest that the proline-rich region may play a role in the subcellular localization and enzymatic function. The transmembrane domain-deleted mutant was indistinguishable from the wild-type enzyme.
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PMID:Deletion of a proline-rich region and a transmembrane domain in fatty acid amide hydrolase. 1041 95

Two unlinked loci, gmdA and bzuA, have previously been identified as being required for the utilization of benzamide as the sole nitrogen source by Aspergillus nidulans. We have cloned each of these genes via direct complementation. The gmdA gene encodes a predicted product belonging to the amidase signature sequence family that displays similarity to AmdS from A. nidulans. However, identity is significantly higher to the amdS gene from Aspergillus niger. The bzuA gene encodes a protein belonging to the cytochrome P450 superfamily and is orthologous to the benzoate para-hydroxylase-encoding gene bphA of A. niger. The bzuA1 mutation prevents the use of benzoate as a carbon source and intracellular accumulation of benzoate results in growth inhibition on benzamide. Northern blot analysis has shown that gmdA expression is subject solely to AreA-dependent nitrogen metabolite repression while bzuA is strongly benzoate inducible and subject to CreA-mediated carbon catabolite repression and a probable inactivation of benzoate induction by glucose. Fluorescence microscopy of a fusion of the N-terminal end of BzuA to green fluorescent protein revealed that this protein localizes to the endoplasmic reticulum.
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PMID:The genes gmdA, encoding an amidase, and bzuA, encoding a cytochrome P450, are required for benzamide utilization in Aspergillus nidulans. 1184 76


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